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A Presentation By
G. Prashanth Kumar
Department of Microbiology & Parasitology,
Faculty of Medicine, International Medical &
Technological University,
Dar-Es-Salaam, Tanzania.
INTRODUCTION:
Bacteria are microscopic organisms. They are also colorless for the
STAIN:
POSITIVE STAINING: - where the actual cells are themselves colored and appear in
a clear background.
(a) Simple staining: A stain which provides color contrast but gives same color to
all bacteria and cells.
Ex: Loeffler’s methylene blue, Polychrome methylene blue, Diluted carbol fuchsin.
Method:
-Aseptically a small sample of the culture is spread over a slide surface.
-The next step is heat fixation to help the cells adhere to the slide surface.
2) Methanol fixation
a) Place air-dried smears in a coplin jar with methanol for one minute.
Alternatively, flood smear with methanol for 1 minute.
b) Drain slides and allow to dry before staining.
SIMPLE STAINING
LOEFFLER’S METHYLENE BLUE:
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ORIGINAL FORMULATION OF DR. GRAM
visualized.
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PRINCIPLE
The Gram Reaction is dependent on permeability of the bacterial cell wall and
cytoplasmic membrane, to the dye-iodine complex.
In Gram positive bacteria, the crystal violet dye –iodine complex combines to form
a larger molecule which precipitates within the cell. Also the alcohol/acetone
mixture which act as decolorizing agent, cause dehydration of the multi-layared
peptidoglycan of the cell wall. This causes decreasing of the space between the
molecules causing the cell wall to trap the crystal violet iodine complex within the
cell. Hence the Gram positive bacteria do not get decolorized and retain primary
dye appearing violet. Also, Gram positive bacteria have more acidic protoplasm and
hence bind to the basic dye more firmly.
In the case of Gram negative bacteria, the alcohol, being a lipid solvent, dissolves
the outer lipopolysaccharide membrane of the cell wall and also damage the
cytoplasmic membrane to which the peptidoglycan is attached. As a result, the dye-
iodine complex is not retained within the cell and permeates out of it during the
process of decolourisation. Hence when a counter stain is added, they take up the
colour of the stain and appear pink.
GRAM POSITIVE BACTERIA
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METHOD CONSISTS OF FOUR COMPONENTS:
Result:
Gram-positive organisms, fibrin, some fungi, keratohyalin, and keratin - purple
By this method fairly good results may be obtained with very short staining
times, which are convenient when only one slide has to be stained.
Flood the slide with crystal or methyl violet stain and allow to act for about 5
seconds.
Tip off the stain and flood the tilted slide with iodine solution and allow to act
for about 5 seconds.
Tip off the iodine and flood the tilted slide
With acetone and allow this to act for only 2 seconds before washing it off with
water from the tap.
Flood the slide with basic fuchsin counter stain and allow it to act for about 5
seconds. Wash off with water, blot and dry.
QUALITY CONTROL
Daily and when a new lot is used, prepare a smear of Escherichia coli (ATCC
25922) and Staphylococcus epidermidis (ATCC 12228)or Staphylococcus aureus
(ATCC 25923). Fix and stain as described.
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MODIFICATION IN GRAM STAINING METHODS
Bartholomew (1962) has pointed out that each variation in the Gram
staining procedure has a definite limit to its acceptability. Any final
result is the outcome of the interaction of all of the possible variables.
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VARIOUS MODIFICATIONS OF GRAM STAINING
1. Kopeloff & Beerman’s Modification :
Primary stain is Methyl violet.
Decolourizer is Acetone/ Acetone-Alcohol mixture.
2. Jensen’s Modification :
Primary stain is Methyl violet.
Decolourizer is Absolute Alcohol.
Counter stain is Neutral Red.
3. Preston & Morrell’s Modification :
Primary stain is Crystal violet.
Decolourizer is Iodine-Acetone.
4. Weigert’s Modification:
Primary stain is Carbol Gentian violet.
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APPLICATIONS OF GRAM STAINING
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ZIEHL-NEELSEN STAINING FOR ACID FAST BACILLI
Modifications :
Kinyoun’s-cold stain
ACID - FAST STAIN BASIC REQUIREMENTS
2. Decolourization with Acid Alcohol : The acid alcohol contains 3% HCl and
95% ethanol or 20% H2 SO4.
Place the dried, heat-fixed smear on a staining rack over the sink. Smears of
sputum should be thin.
Cover the smear with auramine phenol and leave to stain at room temperature
for 10 minutes.
Wash off stain with water from the tap.
Cover the slide with an excess of 1% acid-alcohol and leave to decolorize for 5
minute.
Wash off decolourizer with water from the tap.
Cover the smear with the 0.1% potassium permanganate counter stain
and leave for 15 seconds.
Wash off counter stain with water from the tap.
Dry on heated drier or dry in air. Do not use blotting paper.
Examine the film dry by fluorescence microscopy with a 4 mm objective.
Tubercle bacilli are seen as yellow luminous rods in a dark field. When
detected under low power, the morphology of the bacilli is confirmed
with an oil-immersion objective.
FLUOROCHROME STAIN : MICROSCOPY
A sputum
smear stained
with Auramine
FLUOROCHROME AFB MICROSCOPY
More rapid and sensitive
* Specificity : same with sufficient expérience
Advantages:
• More accurate: 10% more sensitive than light microscopy, with specificity
comparable to ZN staining
• Faster to examine = less technician time
Disadvantages:
• Higher cost and technical complexity, less feasible in many areas
STAINING OF VOLUTIN-CONTAINING ORGANISMS
Corynebacterium
diphtheriae Stained
by Albert’s stain
Green colored bacilli
showing "Chinese-
letter" arrangement
at angles to each
other containing
bluish-black
metachromatic
STAINING OF SPORES
1) Negative staining.
2) Positive staining.
2) Leptospira
3) Borrelia.
These are motile, elongated, flexible and spiral organisms
They are not easily stained The larger spirochetes, e.g. borreliae, stain by ordinary
methods, including Gram’s (giving a negative reaction), Leishman’s and Giemsa’s. The
smaller ones, e.g. treponemes and leptospires, are too thin to be demonstrated by
ordinary stains.. They are best observed in unstained wet films under the dark ground
microscope where their bright appearance and motility draw attention to them.
If a permanent preparation of small spirochetes is required, use may be made of a silver
impregnation method which artificial thickens them with a deposit of silver. The classical
methods of Fontana (for films) and Leavaditi (for sections).
FONTANA’S {SILVER IMPREGNATION} METHOD
Procedure:
Treat the film three times, 30 seconds each time, with the fixative.
Wash off the fixative with absolute alcohol and allow the alcohol to act for 3 min.
Drain off the excess alcohol and carefully burn off the remainder until the film is dry.
Pour on the mordant, heating till steam rises, and allow it to act for 30 seconds.
Wash well in distilled water and again dry the slide.
Treat with ammoniated silver nitrate, heating till steam rises for 30 seconds when the
film becomes brown in colour.
Wash well in distilled water, dry and mount in Canada balsam.
It is essential that the specimen be mounted in balsam under a cover-slip before
examination, as some immersion oils cause the film to fade at once.
The spirochaetes are stained brownish-black on a brownish-yellow background.
FONTANA’S: MICROSCOPY
Leptospires from
a culture stained
by modified
Fontana silver
technique
(3.000×)
examination by
dark- field
microscopy.
FLAGELLA STAINING
zinc free methylene blue. The original stain has now been replaced by
various modifications which are easier to use and give better results.